Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for the detection and characterization of a wide class of molecules. Mass spectrometric analysis is applicable to almost any chemical species capable of forming an ion in the gas phase, and, therefore, provides perhaps the most universally applicable method of quantitative analysis. In addition, mass spectrometry is a highly selective technique especially well-suited for the analysis of complex mixtures comprising different compounds in varying concentrations. Further, mass spectrometric analysis methods can provide detection sensitivities approaching tenths of parts per trillion for some analytes. As a result of these attributes, a great deal of attention has been directed over the last several decades at developing mass spectrometric methods for analyzing biomolecules, such as peptides, proteins, lipids and oligonuceotides in biological samples.
Mass spectrometric analysis involves three fundamental processes: (1) gas phase ion formation, (2) mass analysis whereby ions are separated on the basis of mass-to-charge ratio (m/z), and (3) detection of ions subsequent to separation. The overall efficiency of a mass spectrometer (overall efficiency=(analyte ions detected)/(analyte molecules consumed)) may be defined in terms of the efficiencies of each of these fundamental processes by the equation:EMS=EF×EMA×ED,  (I)wherein:    EMS is the overall efficiency;    EF is the ion formation efficiency=(analyte ions formed)/(analyte molecules consumed during ion formation),    EMA is the mass analysis efficiency=(analyte ions mass analyzed)/(analyte ions consumed during analysis), and    ED is the detection efficiency=(analyte ions detected)/(analyte ions consumed during detection).
Despite wide adoption of mass spectrometry for identifying and characterizing biomolecules, conventional state of the art mass spectrometers have surprisingly low overall efficiencies for these compounds. For example, a quantitative evaluation of the efficiency of a conventional orthogonal injection time-of-flight mass spectrometer (Perseptive Biosystems Mariner) for the analysis of a sample containing a 10 kDa protein yields the following efficiencies, ES=1×10−4, EMA=8×10−7 and ED=9×10−3, providing an overall efficiency of the mass spectrometer of 1 part in 1012. As a result of low overall efficiency, conventional mass spectrometric analysis of biomolecules typically requires large samples and is unable to achieve the ultra-low sensitivity needed for many important biological applications, such as single cell analysis of protein expression and post-translational modification. Therefore, there is a significant need in the technical field for more efficient ion preparation, analysis and detection techniques to enhance the utility of mass spectrometric analysis for target applications in biology and biochemistry.
Over the last decade, new ion preparation methods have revolutionized mass spectrometric analysis of biological molecules. These new ionization methods, which include matrix assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI), provide greatly improved ionization efficiency for a wide range of compounds having molecular weights up to several hundred KiloDaltons. Moreover, MALDI and ESI ionization sources have been successfully integrated with a wide range of mass analyzers, including quadrupole mass analyzers, time-of-flight instrumentation, magnetic sector analyzers, Fourier transform—ion cyclotron resonance instruments and ion traps, to provide selective identification of polypeptides and oligonucleotides in complex mixtures. Mass determination by time-of-flight (TOF) analysis has proven especially well-suited for the high molecular weight biomolecules ionized by ESI and MALDI techniques because TOF has no intrinsic limit to the mass range accessible, provides high spectral resolution and has fast temporal response times. Use of time-of-flight mass analysis with ESI and MALDI ion sources for proteomic analysis is described in detail by Yates in Mass Spectrometry and the Age of the Proteome, Journal of Mass Spectrometry, Vol. 33, 1-19 (1998). As a result of these advances, MALDI-TOF and ESI-TOF have emerged as the two most commonly used ionization techniques for analyzing complex samples containing biomolecules.
Although integration of modern ionization techniques and time-of-flight analysis methods has expanded the mass range accessible by mass spectrometric methods, complementary ion detection methods suitable for time of flight analysis of high molecular weight compounds, including many biological molecules, remain considerably less well developed. Indeed, effective upper limits of mass ranges accessible by state of the art MALDI-TOF and ESI-TOF analysis techniques are limited by the sensitivity of conventional ion detectors for high molecular weight ions. Conventional multichannel plate (MCP) detectors, for example, exhibit sensitivities that decrease with ion velocity, which in the context of time of flight analyzers corresponds to a decrease in sensitivity with increasing molecular weight.
MCP detectors are perhaps the most pervasive ion detectors currently used in ESI-TOF and MALDI-TOF mass spectrometry. These detectors operate by secondary electron emission and typically comprise a parallel array of miniature channel electron multipliers. Typically the channel diameters are in the range of 10 to 100 microns with the lengths of the channels in the neighborhood of 1 mm. Each channel operates as a continuous dynode structure, meaning that it acts as its own dynode resistor chain. A potential of about 1 to 2 kV is placed across each channel. When an energetic, ionized molecule enters the low potential end of the channel and strikes the wall of the channel it produces secondary electrons which are in turn accelerated along the tube by the electric field. These electrons then strike the wall generating more electrons. The process repeats many times until the secondary electrons emerge from the high potential end of the channel. Generally speaking for each molecule which initiates a cascade, 104 electrons emerge from the channel providing significant gain. The electron cascade formed is collected at an anode and generates an output signal. MCP detectors can be made in large area format which is useful for analysis of packets of ions in TOF systems.
A number of limitations of MCP detection systems arise out of the impact-induced mechanism governing the generation of secondary electrons. First, the yield of secondary electrons in a MCP detector decreases significantly as the velocities of ions colliding with the surface decreases. As time-of-flight detectors accelerate all ions to a fixed kinetic energy, high molecular weight ions have lower velocities and, hence, lower probabilities of being detected by MCP detectors. Second, the secondary electron yield of MCP detectors also depends on the composition, size and structure of colliding gas phase ions. Third, it is also established that once a cascade has been initiated within a channel, it is depleted of electrons. Due to the high resistivity of the channel, the time required to replace these electrons is several orders of magnitude larger (milliseconds) than the duration of the TOF measurement (microseconds). Thus, for a single TOF event a channel is rendered inactive (“deadtime”) after a single cascading event, thus each successive packet of ions impinging on the detector has fewer and fewer active channels available to it.
As apparent to those skilled in the art of mass spectrometry, these limitations impact the utility of MCP detectors for certain mass spectrometry applications by hindering quantitative analysis of samples containing high molecular weight biopolymers. Accordingly, a need currently exists for ion detectors for mass spectrometry that do not exhibit decreasing sensitivities with increasing molecular weight and that do not have sensitivities dependent on the composition and structure of gas phase ions analyzed.
U.S. Patent Publication No. 2007/0023621, published Feb. 1, 2007, U.S. Patent Publication No. 2009/0321633, published Dec. 31, 2009, and U.S. Patent Publication No. 2010-0320372, published Dec. 23, 2010, disclose detectors for mass spectrometry having a nano- or microstructured membrane geometry. In these systems, impact of ions on a receiving surface of an electrically biased semiconductor membrane generates field emission from the membrane. The references provide modeling data and experimental results showing that measurement of field emission from the membrane as a function of time provides a means for detecting and analyzing ions, for example, by determination of the flight times of ions separated on the basis of mass to charge exiting a time of flight analyzer.
It will be appreciated from the foregoing that there is currently a need in the art for methods, systems and devices for detecting and analyzing molecules having large molecular masses. Specifically, detection methods and systems providing sensitive detection of large molecular mass molecules are needed that are capable of effective integration with conventional mass spectrometry systems, such as TOF analysis systems. Sensors and analyzers are needed for mass spectrometry applications that do not exhibit decreases in sensitivity as a function of molecular mass, and that are capable of fast readout and good temporal resolution.